Promoter to IL-18BP, its preparation and use

ABSTRACT

The present invention relates to the promoter of interleukin-18 binding protein (IL-18BP), to its preparation and use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase Entry Under 35 U.S.C. §371of International Application No. PCT/2003/000815, filed Oct. 9, 2003,which designated the U.S., and which claimed benefit under 35 U.S.C. 119of the Israeli Patent Application No. 152232, filed in English languageon 10 Oct. 2002.

FIELD OF THE INVENTION

The present invention relates to the promoter of interleukin-18 bindingprotein (IL-18BP), to its preparation and use.

BACKGROUND OF THE INVENTION

Cytokine binding proteins (soluble cytokine receptors) are usually theextracellular ligand binding domains of their respective cell surfacecytokine receptors. They are produced either by alternative splicing orby proteolytic cleavage of the cell surface receptor. These solublereceptors have been described in the past: for example, the solublereceptors for IL-6 and IFNγ (Novick et al. 1989), TNF (Engelmann et al.1989 and Engelmann et al. 1990), IL-1 and IL-4 (Maliszewski et al. 1990)and IFNα/β (Novick et al. 1994, Novick et al. 1992). Onecytokine-binding protein, named osteoprotegerin (OPG, also known asosteoclast inhibitory factor—OCIF), a member of the TNFR/Fas family,appears to be the first example of a soluble receptor that exists onlyas a secreted protein (Anderson et al. 1997, Simonet et al. 997, Yasudaet al. 1998).

An interleukin-18 binding protein (IL-18BP) was affinity purified, on anIL-18 column, from urine (Novick et al. 1999). IL-18BP abolishes IL-18induction of IFNγy, and IL-18 activation of NF-kB in vitro. In addition,IL-18-BP inhibits induction of IFNγ in mice injected with LPS. TheIL-18BP gene was localized to the human chromosome 11, and no exoncoding for a transmembrane domain could be found in the 8.3 kb genomicsequence comprising the IL-18BP gene. Four isoforms of IL-18BP generatedby alternative mRNA splicing have been found in humans so far. They weredesignated IL-18BP a, b, c, and d, all sharing the same N-terminus anddiffering in the C-terminus (Novick et al 1999). These isoforms vary intheir ability to bind IL-18 (Kim et al. 2000). Of the four, humanIL-18BP (hIL-18BP) isoforms a and c are known to have a neutralizingcapacity for IL-18. The most abundant IL-18BP isoform, the splicedvariant isoform a, exhibits a high affinity for IL-18 with a rapidon-rate and a slow off-rate, and a dissociation constant (Kd) ofapproximately 0.4 nM (Kim et al. 2000). IL-18BP is constitutivelyexpressed in the spleen (Novick 1999), and circulates at plasmaconcentrations of 2.5 ng/ml (Novick et al. 2001). The residues involvedin the interaction of IL-18 with IL-18BP have been described through theuse of computer modelling (Kim et al. 2000) and based on the interactionbetween the similar protein IL-1β with the IL-1R type I (Vigers et al.1997). According to the model of IL-18 binding to the IL18BP, the Gluresidue at position 42 and Lys residue at position 89 of IL-18 have beenproposed to bind to Lys-130 and Glu-114 in IL-18BP, respectively (Kim etal. 2000).

As mentioned, IL-18 induces IFNγ which, in turn, was recently reportedto induce IL-18BPa mRNA generation in vitro (Muhl et al 2000).Therefore, IL-18BPa could serve as a “shut off” signal, terminating theinflammatory response.

IL-18BP is significantly homologous to a family of proteins encoded byseveral Poxviruses (Novick et al. 1999, Xiang and Moss 1999). Inhibitionof IL-18 by this putative viral IL-18BP may attenuate the inflammatoryantiviral Th1 response. Serum IL 18BP is significantly elevated insepsis, indicating its role in regulating immune responses in vivo(Novick et al. 2001). Indeed, IL-18BP is induced by IFNγ in variouscells, suggesting that it serves as a negative feedback inhibitor of theIL-18-mediated immune response (Mughl et al. 2000)

Preliminary results indicate that IL-18BP mRNA is detected inleukocytes, colon, small intestine, prostate and particularly in spleencells (Novick et al. 1999). The component cells of the spleen consist ofmacrophages, lymphocytes, and plasma cells with additional cells derivedfrom the circulation.

The activity of elements that control transcription, promoter andenhancers vary considerably among different cell types. Promoters andenhancers consist of short arrays of DNA sequences that interactspecifically with cellular proteins involved in transcription (reviewedin Dynan and Tjian 1985, McKnight and Tjian 1986, Sassone-Corsi andBorreli 1986 and Maniatis et al 1987). The combination of differentrecognition sequences and the amounts of the cognate transcriptionfactors determine efficiency with which a given gene is transcribed in aparticular cell type. Many eukaryotic promoters contain two types ofrecognition sequences: the TATA box and the upstream promoter elements.The TATA box, located 25-30 bp upstreaqm of the transcription initiationsite, is thought to be involved in directing RNA polymerase II to beginRNA synthesis at the correct site. In contrast, the upstream promoterelements determine the rate at which transcription is initiated.Enhancer elements can stimulate transcription up to 1000-fold fromlinked homologous or heterologous promoters. However unlike upstreampromoter elements, enhancers are active when placed downstream from thetranscription initiation site or at considerable distance from thepromoter. Many enhancers of cellular genes work exclusively in aparticular tissue or cell type (reviewed by Voss et al. 1986, Maniatiset al. 1987). In addition some enhancers become active only underspecific conditions that are generated by the presence of an inducer,such as a hormone or metal ion (reviewed by Sassone-Corsi and Borrelli1986 and Maniatis 1987). Because of these differences in cellspecificities of cellular enhancers, the choice of promoter and enhancerelements to be incorporated into a eukaryotic expression vector will bedetermined by the cell types in which the recombinant gene is to beexpressed. Conversely, the use of a prefabricated vector containing aspecific promoter and cellular enhancer may severely limit the celltypes in which expression can be obtained.

Many enhancer elements derived from viruses have a broader host rangeand are active in a variety of tissues, although significantquantitative differences are observed among the different cell typees.For example, the SV40 early enhancer is promiscuously active in manycell types derived from a variety of mammalian species, and vectorsincorporating this enhancer have consequently been used (Dijkema et al.1985). Two other enhancer/promoter combinations that are active in abroad range of cells are derived from the long repeat (LTR) of the Roussarcoma virus genome (Gorman et al 1982b) and from human cytomegalovirus(Boshart et al. 1985).

SUMMARY OF THE INVENTION

The invention relates to a DNA sequence encoding the human IL-18BPpromoter (SEQ ID NO: 1), or a fragment such as that in SEQ ID NOS 2 or3, or a functional derivative thereof wherein the 3′ end of said DNAsequence or fragment thereof comprises one or more nucleotides from the5′ end of SEQ ID NO: 5.

More specifically, a derivative according to the invention can be theDNA of the invention mutated at one or more AP1 sites present in asilencer element present in the sequence, and the DNA sequence mayfurther containing a gene operatively linked to the IL-18BP promoter.

In one aspect of the invention, the gene may encode e. g. IL-18BP or aheterologous protein such as luciferase, interferon-beta, TNF,erythropoietin, tissue plasminogen activator, granulocyte colonystimulating factor, manganese-superoxide dismutase, an immunoglobulin,or fragment thereof, growth hormone, FSH, hCG, IL-18, hsLDLR and TNFreceptor binding proteins.

The invention provides a vector comprising a DNA sequence sequenceencoding the human IL-18BP promoter, a host cell comprising the vectore.g. CHO, WISH, HepG2, Cos, CV-1, HeLA, and Hakat U937 cells, and amethod for the production of a recombinant protein comprising culturingthe host cell and isolating the recombinant protein produced.

In addition, the invention provides a recombinant virus vector whichcomprises a portion of the virus genome, a DNA fragment encoding a geneof interest and a DNA fragment comprising a DNA sequence encoding thehuman IL-18BP promoter. More specifically the virus portion can be e.g.an adeno associated virus, and a retrovirus such as HIV, HFV, MLV, FIVand VSV.

Also the present invention provides a method of regulating cell specificexpression of a gene of interest, comprising transducing a targetmammalian cell with the virus vector of the invention in a target cellsuch as an hematopoietic stem cell, and a monocyte. The gene of interestcan be e.g. a protein conferring resistance to HIV infection. Regulatingcell specific expression of a gene of interest can be used in thetreatment of e. g. HIV infection, the treatment of hematopoieticdisorders such as SCID, chronic granulomatous disease and thalasemia.

The invention further provides a method of gene therapy for thetreatment of a disease in an individual exhibiting elevated IFNγ in abody tissue, comprising the administration of an effective amount of thevirus vector of the invention, optionally further comprisingadministration of IL-6 and/or TNF-a and/or IRF and or C/EBPβ factors.

In another aspect the invention relates to transgenic mice harbouringthe DNA sequence encoding a DNA sequence of the invention.

In addition the invention teaches the use of a DNA sequence encoding thehuman IL-18BP promoter (SEQ ID NO:1), or a fragment or a functionalderivative thereof wherein the 3′ end of said DNA sequence or fragmentthereof comprises one or more nucleotides from the 5′ end of SEQ ID NO:5, in the manufacture of a medicament for the treatment of a disease.

Also, the invention provides a pharmaceutical composition comprising atherapeutically effective amount of a DNA sequence encoding the humanIL-18BP promoter (SEQ ID NO:1), or a fragment or a functional derivativethereof wherein the 3′ end of said DNA sequence or fragment thereofcomprises one or more nucleotides from the 5′ end of SEQ ID NO: 5.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of the promoter region of theIL-18BP gene, including the 5 regulatory elements.

FIG. 2 shows the Kinetics of IL-18BP induction and synergy with TNFα andIL-6. (A) IFNγ induces IL-18BP in a dose and time-dependent manner inhuman WISH cells. Cells were incubated with the indicated concentrationsof IFNγ for 24 and 48 h. (B) Synergistic effects of TNFα IL-6 and theircombination on IFNγ-induced IL-18BP. HepG2 cells were incubated with theindicated combinations of IFNγ (100 U/ml), TNFα. (20 ng/ml) and IL-6(300 U/ml). Induction of IL-18BP by each combination was significantlyhigher (p<0.05) then induction by IFNγ alone. Data are mean SD (n=3, forA. n=4, for B).

FIG. 3. shows a schematic representation of the conserved exon-intronorganization of the human and mouse IL-18BP genes. The human IL-18BPagene was compared with the mouse IL-18BPd gene. Exons are indicated.Transcription start site, translation start site (ATG), stop codon(Stop) and the polyadenylation signal (PAS) are indicated for the humanIL-18BPa gene.

FIG. 4 shows that the induction of IL-18BP by IFNγ is at thetranscriptional level and depends on de novo protein synthesis. (A)semi-quantitative RT-PCR of IL-18BP mRNA from HepG2 cells that werepre-incubated with actinomycin D (1 μg/ml, 30 min), washed and incubatedwith IFNγ (100 U/ml) for the indicated times. RT-PCR of β actin mRNA isshown as a control (B) semi-quantitative RT-PCR of IL-18BP mRNA fromHepG2 cells that were pre-incubated with cycloheximide (20 μg/ml) andIFNγ (100 U/ml) for the indicated times.

FIG. 5 shows the basal and IFNγ-induced activity of luciferase reportervectors carrying the human IL-18BP promoter. Insert size, extending fromthe transcription start site (+1) is given in parentheses. Circlednumbers represent the various response elements: 1. GAS. 2. IRF-E. 3.C/EBP-E (2 sites). Scilencer 5. Distal enhancer. Filled squares depictmutation in a specific response element. HepG2 cells were co-transfectedwith the indicated reporter vector and pSV40 βGAL. All luciferase valueswere normalized to βgalactosidase activity. (A) luciferase activity inextracts of un-induced cells relative to that of cells transfected withpGL3—Basic vector. (B) luciferase activity in cells transfected withselected vectors and induced with IFNγ. Fold induction is over basalactivity as given in (A).

FIG. 6 shows that IRF-1 is essential for IL-18BP expression in mice.Serum IL-18BP of C57B1/6 IRF-1^(−/−) and control C57 B1/6 mice that wereinjected intraperitoneally with murine IFNγ (53,000 u/mouse). Mice werebled before injection and 24 h post injection. Serum IL-18BP wasdetermined by ELISA. Data are mean±SE (n=6 for each group). Thedifferences between serum IL-18BP in control and IRF-deficient mice, aswell as the induction of IL-18BP in control mice were statisticallysignificant (p<0.05).

FIG. 7 shows the role of IRF-1 and C/EBPβ in IL-18BP gene induction andtheir association. (A) electrophoretic mobility shift assay (EMSAExample 18) of dsDNA probes corresponding to bases -33 to -75 (IRF-E,left panel) and -8 to -55 (GAS, right panel). HepG2 cells were treatedwith IFNγ for the indicated times and nuclear extracts were allowed toreact with the IRF-E or GAS probes. Shifted bands are indicated byfilled arrowheads. The GAS complex was also subjected to super shiftwith the indicated antibodies. The super shifted band is indicated by anopen arrowhead. (B) semi-quantitative RT-PCR of IL-18BP mRNA from HepG2cells that were transfected with the indicated combinations of IRF-1 orC/EBPβ expression vectors. Where indicated, IFNγ was added and cellswere harvested 5 h later. Values were normalized to β actin mRNA. (C)luciferase activity in cells transfected with the luciferase reportervector pGL3(1272), containing the complete IL-18BP promoter, togetherwith the indicated concentration of pCDNA3-IRF-1 (circles) and 1 μg/10⁶cells of pCDNA3-C/EBPβ. Alternatively, cells were transfected with theindicated concentration of pCDNA3-C/EBPβ (squares) and 0.1 μg/10⁶ cellsof pCDNA3-IRF-1. Luciferase activity was normalized by the βGalactivity. (D) immunoblots of nuclear and cytoplasmic extracts (seeExample 17 for preparation of extracts) of cells treated with IFNγ (100U/ml, 2 h). Extracts were immunoprecipitated (IP) and immunoblotted (IB)with the indicated antibodies.

FIG. 8 shows the factors binding to the promoter of IL-18BP upon IFNγinduction (A) EMSA with the proximal C/EBPβ E and whole cell extractsfollowing treatment with IFNγ. Where indicated, the extracts were supershifted with the indicated antibodies. (B) EMSA with a probecorresponding to the distal enhancer and whole cell extracts followingtreatment with IFNγ. Where indicated, the extracts were super shiftedwith the indicated antibodies, with or without competition with ds DNAcorresponding to the proximal half of the probe. Shifted bands areindicated by filled arrowheads and super shifted band are indicated byopen arrowheads.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the promoter of human IL-18BP. Thispromoter drives the constitutive expression of IL-18BP in particularcells, for example in monocytes and the IFNγ mediated induction ofIL-18BP expression in many cells. The promoter of human IL-18BP iscapable of directing the constitutive and IFNγ induced expression of aheterologous protein.

The invention relates to a DNA sequence encoding the human IL-18BPpromoter (SEQ ID NO:1), or a fragment or a functional derivative thereofwherein the 3′ end of said DNA sequence or fragment thereof comprisesone or more nucleotides from the 5′ end of SEQ ID N: 5.

IL-18BP mRNA is detected in leukocytes, colon, small intestine, prostateand mainly in spleen cells (Novick et al. 1999). In one of the examplesbelow it was shown that IL-18 protein is constitutively expressed inmonocytes.

IL-18BP expression was found to be induced by IFNγ, not only inmonocytes but also in many different cells and that this induction couldbe further enhanced by the addition of IL-6 and TNFα.

De novo protein synthesis was found to be essential for IL-18BP geneactivation by IFNγ.

The transcription start site of human IL-18BPa mRNA was determined by 5′RACE.

The 3′ end mRNA of the Zn finger protein located upstream of the IL-8BPgene was found thereby limiting the potential upstream regulatorysequence of the IL-18BPa to 1601 bases upstream of base 1.

Six regulatory elements (FIG. 1) were identified within this region(from the transcription proximal to the transcription distal): 1- Agamma-activated sequences (GAS) at bases -24 to -32, 2- An IRF-1,2response element (IRF-E) spanning bases -57 to -69, 3- and 4- two C/EBPβresponse elements at bases -309 to -322 and -621 to -634, 5- a scilencerat residues -625 to -1106 and 6- an enhancer element spanning bases-1106 to -1272. A series of luciferase reporter vectors with progressivetruncations at the 5′end of the 1601 bp fragment were tested in HepG2cells (a human hepatocellular carcinoma line). The 1272 kb region setforth in SEQ ID NO: 1 direct both, a basal expression seen in sometissues and cell types, as well as induction by IFNγ. Testing promoteractivity on successive truncated DNA fragments within this regiondemonstrated that a 122 bp DNA fragment, proximal to the transcriptionstart site set forth in SEQ ID NO: 3 comprises the minimal promoter.This minimal promoter is also inducible. However, other regulatorysequences upstream of this minimal promoter did contribute to the extentof induction. A DNA fragment of 635 bp containing in addition to theIRF-1 and GAS elements two C/EBPβ elements, set forth in SEQ ID NO: 2,was found to confer maximal induction of luciferase expression by IFNγ.

In-vivo experiments carried out with IRF-1-deficient mice confirmed theimportance of IRF-1 as a mediator of basal as well as IFNγ-inducedexpression of IL 18BP.

It was found that upon IFNγ induction, the expression of the IRF-1factor is induced and the factor is complexed to C/EBPβ which isconstitutively present in the cells. The complex binds to the proximalGAS promoter element and its adjacent IRF-E promoter element.

The enhancer present at the transcription site distal end was found tointeract with the basal promoter through IRF-1.

The present invention relates to the IL18BP promoter of SEQ ID NO:1 or afragment thereof and methods for regulating gene expression. Moreparticularly, the present invention relates to the isolated DNAsequences of IL-18BP 1272 bp (SEQ ID NO:1) or a fragment thereof suchas, 635 bp (SEQ ID NO: 2) and 122 bp (SEQ ID NO: 3) which are capable ofdirecting gene expression.

This IL-18BP promoter region has been cloned and sequenced andcorresponds to nucleotides in the 1272 bp upstream of the transcriptionstart site of IL-18BP (SEQ. ID. NO: 1).

The present invention encompasses the entire IL-18BP promoter (SEQ IDNO:1), but also DNA sequences comprising a fragment thereof (SEQ IDNO:2, SEQ ID NO:3), capable of directing gene transcription, andtherefore ultimately gene expression, and can be used with otherportions of the IL18BP promoter region or alternatively withheterologous promoters or heterologous promoter elements to control genetranscription. This promoter or fragment thereof is capable of inductionby IFNγ. Such induction can be further enhanced by overexpression ofIRF-1 and/or C/EBPβ and/or by treatment with IL-6 and/or TNFα

Functional derivatives of promoter set forth in SEQ ID NO:1, or fragmentthereof such as SEQ ID NO:2 or SEQ ID NO:3 are mutants wherein 1 to 10,preferably 1 to 5, more preferably 1 nucleotide is replaced withanother, or is deleted and which are capable of directing geneexpression and IFNγ induction.

The DNA sequences of the present invention comprising a IL-18BP promoter(SEQ ID NO: 1) or a fragment thereof such as that in SEQ ID NO:2 or SEQID NO:3, can be isolated using various methods known in the art. Atleast three alternative principal methods may be employed:

(1) the isolation of the DNA sequence from genomic DNA which containsthe sequence; (2) the chemical synthesis of the DNA sequence; and (3)the synthesis of the DNA sequence by polymerase chain reaction (PCR).

In the first approach, a human genomic DNA library can be screened inorder to identify a DNA sequence comprising a IL-18BP promoter orIL-18BP promoter element.

In the second approach, a DNA sequence comprising a IL-18BP promoter ora IL-18BP promoter element can be chemically synthesized. For example, aDNA sequence comprising a IL-18BP promoter region or a IL-18BP promotercan be synthesized as a series of 100 base oligonucleotides that canthen be sequentially ligated (via appropriate terminal restrictionsites) so as to form the correct linear sequence of nucleotides.

In the third approach, a DNA sequence comprising a IL-18BP promoterregion or a IL-18BP promoter can be synthesized using PCR. Briefly,pairs of synthetic DNA oligonucleotides at least 15 bases in length (PCRprimers) that hybridize to opposite strands of the target DNA sequencecan be used to enzymatically amplify the intervening region of DNA onthe target sequence. Repeated cycles of heat denaturation of thetemplate, annealing of the primers and extension of the 3′-termini ofthe annealed primers with a DNA polymerase results in amplification ofthe segment defined by the 5′ ends of the PCR primers. See, U.S. Pat.Nos. 4,683,195 and 4,683,202.

The IL-18 BP promoter of the invention was shown to be capable ofconferring basal expression and also induced expression by IFNγ of anheterologous gene. Thus, the promoter of IL-18BP have both basal andinducible activity.

While the nucleotide sequence of the promoter is set forth in SEQ. ID.NO.1 or to fragments thereof having promoter activities and reference ismade to such sequence in the specification, it is recognized thatnucleotide derivatives can be made which do not affect the promoter orpromoter element function. These modified nucleotide sequences may beprepared, for example, by mutating the nucleotide sequence so that themutation results in the deletion, substitution insertion, inversion oraddition of one or more nucleotides using various methods known in theart. For example, the methods of site-directed mutagenesis described inTaylor, J. W. et al., Nucl. Acids Res. 13, 8749-8764 (1985) and Kunkel,J. A., Proc. Natl. Acad. Sci. USA 82, 482-492 (1985) may be employed. Inaddition, kits for site-directed mutagenesis may be purchased fromcommercial vendors. For example, a kit for performing site-directedmutagenesis may be purchased from Amersham Corp. (Arlington Heights,Ill.). The present invention encompasses DNA containing sequences atleast 50% identical and preferably 75% identical and more preferably 90%identical to SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively,provided that the promoter activity is retained and/or enhanced. Onesuch derivative e.g. in SEQ ID NO:1 is that mutated in one or all of thethree AP1 sites present in the silencer region.

The nucleotide sequence comprising the promoter of IL-18BP, a fragmentthereof and/or a derivative thereof can be operably linked to the codingregion of any gene of interest to express that gene in an appropriatehost cell. By operably linked is intended operably linked for promoterand elements. For expression of a gene of interest, it is preferred thatthe entire IL-18BP promoter in SEQ. ID. NO.1: or a fragment thereof suchin SEQ ID 2 or SEQ ID NO:3 or a derivative thereof is operably linked tothe gene of interest. As shown below in the example section, the IL-18BPpromoter, or a fragment thereof such as that in SEQ ID NO:2 or in SEQ IDNO:3 is capable of directing the expression of heterologous genes. Theexpression of homologous genes with the promoter of the invention isalso contemplated.

The promoter may further contain an intron, for example the first intronof IL-18BP.

An “operably linked” IL-18BP promoter or promoter element will directthe transcription of a nucleic acid molecule joined in proper readingframe. With regard to heterologous promoters, the promoters and elementsof the invention are operably linked when they control the function ofsuch heterologous promoters.

As noted above, the IL-18BP promoter, a fragment thereof and aderivative sequences thereof of the present invention can be utilized toexpress any gene of interest. Typically, an expression vector is usedfor this purpose. Thus, the present invention further concernsexpression vectors comprising an isolated DNA sequence capable ofdirecting gene expression which comprises a IL-18BP promoter or afragment thereof or a derivative thereof. The expression vectorspreferably contain an IL-18BP promoter a fragment thereof or derivativethereof having a nucleotide sequence corresponding to SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:3, respectively or fragments thereof and/orderivatives. Also preferred are expression vectors further comprising ahomologous or heterologous gene operatively linked to the IL-18BPpromoter or a fragment thereof and/or a derivative thereof modifiednucleotide sequence thereof.

Expression vectors of utility in the present invention are often in theform of “plasmids”, which refer to circular double stranded DNAs which,in their vector form, are not bound to the chromosome. However, theinvention is intended to include such other forms of expression vectorswhich serve equivalent functions and which become known in the artsubsequently hereto.

Expression vectors useful in the present invention typically contain anorigin of replication, a IL-18BP promoter located in front of (i.e.,upstream of) the gene of interest, transcription termination sequencesand the remaining vector. The expression vectors can also include otherDNA sequences known in the art, for example, stability leader sequenceswhich provide for stability of the expression product, secretory leadersequences which provide for secretion of the expression product,sequences which allow expression of the structural gene to be modulated(e.g., by the presence or absence of nutrients or other inducers in thegrowth medium), marking sequences which are capable of providingphenotypic selection in transformed host cells, and sequences whichprovide sites for cleavage by restriction endonucleases. Thecharacteristics of the actual expression vector used must be compatiblewith the host cell which is to be employed. An expression vector ascontemplated by the present invention is at least capable of directingthe transcription, and preferably the expression, of the gene ofinterest dictated by the IL-18BP promoter region or IL-18BP promoter ora modified nucleotide sequence thereof. Suitable origins of replicationinclude, for example, that of the Simian virus 40 (SV40). Suitabletermination sequences include, for example, that of the Simian virus 40(SV40). The promoter of the invention can be employed for the expressionof virtually any gene of interest, for example, those encodingtherapeutic products such as interferon-beta, TNF, erythropoietin,tissue plasminogen activator, granulocyte colony stimulating factor,manganese-superoxide dismutase, an immunoglobulin, or fragment thereof,growth hormone, hsLDLR, FSH, hCG, IL-18, TNF receptor binding proteinsand IL-18 binding proteins. All of these materials are known in the artand many are commercially available.

Suitable expression vectors containing the desired coding and controlsequences may be constructed using standard recombinant DNA techniquesknown in the art, many of which are described in Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989).

The present invention additionally concerns host cells containing anexpression vector comprising an isolated DNA sequence capable ofdirecting gene expression which comprises a IL-18BP promoter region or aIL-18BP promoter or modified nucleotide sequences thereof. Preferably,the IL-18BP promoter region has the nucleotide sequence corresponding to1272 bp upstream of the transcription start site of IL-18BP set forth inSEQ D) NO:1, or a fragment thereof such as the nucleotide sequencecorresponding to 635 bp upstream of the transcription start site setforth in SEQ ID NO: 2 or a fragment thereof such as the nucleotidesequence of 122 bp upstream of the transcription start site set forth inSEQ ID NO:3. Also preferred are host cells containing an expressionvector further comprising a homologous or heterologous gene operativelylinked to the IL-18BP promoter region or the IL-18BP set forth in SEQ IDNO:1 or a fragment thereof. Suitable host cells include, for example,human HeLa cells or African Green Monkey cells CV-1 and COS-1, CHOcells, HepG2, WISH cells, Hakat U937 etc.

Preferred are host cells containing receptors for IFNγ, which allowinduction of the IL-18BP promoter and therefore enhanced expression ofthe gene of interest.

Expression vectors may be introduced into host cells by various methodsknown in the art. For example, transfection of host cells withexpression vectors can be carried out by the calcium phosphateprecipitation method. However, other methods for introducing expressionvectors into host cells, for example, electroporation, biolistic fusion,liposomal fusion, nuclear injection and viral or phage infection canalso be employed.

Once an expression vector has been introduced into an appropriate hostcell, the host cell can be cultured and the polypeptide encoded by thegene of interest can be isolated. Alternatively once an expressionvector has ben introduced into in an appropriated host cell, the cellcan be cultured and after reaching a desired cell density, the cells canbe stimulated with IFNγ and the polypeptide encoded by the gene ofinterest can be isolated.

Host cells containing an expression vector which contains a DNA sequencecoding for a gene of interest may be identified using various methodsknown in the art. For example, DNA-DNA hybridization, assessing thepresence or absence of marker gene functions, assessing the level oftranscription as measured by the production of mRNA transcripts of thegene of interest in the host cell, and detecting the gene productimmunologically can be employed.

The DNA sequences of expression vectors, plasmids or DNA molecules ofthe present invention may be determined by various methods known in theart. For example, the dideoxy chain termination method as described inSanger et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977), or theMaxam-Gilbert method as described in Proc. Natl. Acad. Sci. USA 74,560-564 (1977) may be employed.

It should be understood specific nucleotides or regions within theIL-18BP promoter may be identified as necessary for regulation. Theseregions or nucleotides may be located by fine structural dissection ofthe elements, and can be studied by experiments which analyze thefunctional capacity of promoter mutants. For example, single base pairmutations of promoter elements or progressive deletions, as suchemployed in the example section below, can be generated utilizing PCR.In this fashion, a number of mutated promoter regions or deletionconstructs are amplified, and then cloned back into reporter constructsand evaluated with transfection and luciferace assay techniques (as setforth in the example section below). These amplified fragments can becloned back into the context of the IL-18BP promoter and also into theheterologous promoter constructs. In this fashion, the exact nucleotidesequences that are important in directing gene transcription areidentified.

This analysis will also identify nucleotide changes which do not effectpromoter function, or which may increase promoter function. Thus,functional derivative promoters and promoter elements can also beconstructed.

Functional analysis of the promoter region or promoter can befacilitated by footprint and gel-shift studies. Knowledge of the exactbase pairs important in mediating binding of proteins provides evidenceof bases important in mediating the transcriptional response.

The invention therefore further encompasses the base pairs important inDNA-protein interaction. Such base pairs can be elucidated. Genomicfragments containing the areas of interest can be employed in in vitrofootprinting experiments [Galas et al., Nucleic Acids Res. 9, 6505-6525(1981)]. Isolated restriction fragment can be radiolabled andsubsequently incubated with nuclear extracts made with establishedtechniques from cells expected to contain DNA binding proteins whichwill bind to the fragment [for example, Dignam et al., Nucleic AcidsRes. 11, 1475-1489 (1983)]. Labeled DNA fragments are incubated with thenuclear extracts, digested with DNAse I, and electrophoresed on adenaturing polyacrylamide gel. DNA binding proteins in the cell extractbind to their recognition sequence contained in the labeled restrictionfragment, and protect the DNA from digestion by the DNAse. Regions ofprotection delineate the binding site. Maxam and Gilbert sequencingreactions of the fragment can be used as a marker to define thenucleotides protected from DNAse digestion.

The invention is further drawn to the identification andcharacterization of trans-acting factors which interact with thepromoter or promoter elements. Cis-acting regulatory sequences serve asbinding sites for proteins which are termed transacting factors (TAF)[Dynan W. S., Tjian T. Nature 316, 774-778 (1985); Maniatis, T. et al.,Science 236, 1237-1245 (1987)]. Each gene is presumed to bind one ormore proteins at each of its regulatory sequences, and these proteinsinteract with one another and RNA polymerase II in a fashion thatcontrols transcription.

TAFs have been identified in nuclear extracts by their ability to bindto and retard electrophoretic mobility of cis-acting sequence DNAfragments [Dignam, J. D. et al., Nucleic Acids Res. 11, 1475-1489(1983); Dynan, W., Cell 58, 1-4 (1989); Fletcher, C. et al., Cell773-781 (1987); Scheidereit, C. et al., Cell 51, 783-793 (1987)].

The cis-acting sequences are useful in gel retardation assays todetermine binding activity in nuclear extracts. The technology for gelshift assays is described in the literature and includes many of thesame reagents used in footprint experiments [Fried, M. et al., NucleicAcids Res. 9, 6505-6525 (1981); Revzin, A., Biotechniques 7, 346-355(1989); Strauss, F. A. et al., Cell 37, 889-901 (1984)]. Either 32P-labeled restriction fragments or annealed pairs of complementaryoligos are incubated with nuclear extracts and poly d(I-C) in a bindingbuffer, and the products of this reaction electrophoresed on anon-denaturing polyacrylamide gel. The location of the DNA fragment onthe gel as determined with autoradiography is retarded in cases whereprotein has bound to the DNA. The extend of the retardation is arelative function of the size of the protein.

The binding proteins so identified can then be purified and ultimatelycloned using known techniques.

The promoter of IL-18BP also find use in transgenic studies. Transgenicmice provide a powerful genetic model for the study of a number of humandiseases including cancer. They have also provided an important in vivomethod for studies of gene regulation that have confirmed and extendedobservations made with transfection reporter gene (e.g. luciferase)experiments [Palmiter, F. L. et al., Ann. Rev. Genet. 20, 465-499(1986)]. Studies aimed at dissecting the signals allowing developmentalrelation of gene expression can rarely be performed in cell culturemodels and is probably best studied with a transgenic model. This typeof experiment is possible because of the remarkable conservation betweenspecies of regulatory sequences, such that human regulatory signals areaccurately interpreted by the mouse transcription machinery.

Constructs expressed in transgenic mice could therefore provide muchinformation about the regulation of the IL-18BP gene.

Transgenic mice can be made by methods known in the art. The most widelyused method through which transgenic animals have been produced involveinjecting a DNA molecule into the male pronucleus of a fertilized egg[Brinster et al., Cell 27, 223 (1981); Costantini et al., Nature 294,982 (1981); Harpers et al., Nature 293, 540 (1981); Wagner et al., Proc.Natl. Acad. Sci. USA 78, 5016 (1981); Gordon et al., Proc. Natl. Acad.Sci. USA 73, 1260 (1976)].

Once the DNA molecule has been injected into the fertilized egg cell,the cell is implanted into the uterus of recipient female and allowed todevelop into an animal. Thus, all of the cells of the resulting animalshould contain the introduced gene sequence.

The resulting transgenic mice or founders can be bred and the offspringanalyzed to establish lines from the founders that express thetransgene. In the transgenic animals, multiple tissues can be screenedto observe for gene expression. RNA studies in the various mouse lineswill allow evaluation of independence of the integration site toexpression levels of the transgene. See, Hogan, B. et al., Manipulatingthe mouse embryo: a laboratory manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1986).

The IL-18BP promoter and promoter elements may also provide a usefulmeans for carrying out gene therapy.

“Gene therapy” is the administration of genetic material to modify ormanipulate the expression of a gene product to alter biologicalproperties of living cells for therapeutic use.

The cells can be allogeneic or autologous. Cells may be modified ex-vivofor subsequent administration to the subject or altered in vivo by genetherapy products given directly to the subject.

For the most part, constructs comprising the IL-18BP promoter orfragment thereof and/or a derivative thereof will be utilized to targetgene expression in those cells when the IL18BP gene is normallyexpressed, for example mononuclear cells. Any means available in the artfor transfer of the constructs into animals, including humans, can beutilized. This includes viral vectors, particularly retroviral vectors(see, for example, Zweibel et al, Science 243, 220 (1989), and thereferences cited therein), as well as other methods.

Recombinant AAV vectors have been shown quite promising for therapeuticgene delivery in liver and skeletal muscle (Snyder et al. 1997, Murphyet al. 1997, Song et al. 1998, Snyder et al. 1999, Herzog et al. 1997).Mice generated by disrupting the clotting factor IX gene exhibit severebleeding disorder and closely resemble the phenotype seen in hemophiliaB patients. It has been reported (Wang et al. 1999) that a singleintraportal injection of a recombinant adeno-associated virus (AAV)vector encoding canine factor IX cDNA under the control of aliver-specific enhancer/promoter leads to a long-term and completecorrection of the bleeding disorder.

Retroviral vectors, derived from oncoretrovirus such as the murineleukemia virus (MVL), have been the most widely used vectors for genetransfer because the vector genome integrates into the chromosomes oftarget cells, resulting in stable expression of transgenes (I. M. Vermaand N. Somnia. Nature 389, 239 (1997) however these vectors were provento be good especially for dividing cells. Letiviruese vectors such asHIV vectors are being currently used for nondividing cells Mioshi et al.Science 1999 283: 682-686. The ability of lentiviruses to infectnondividing cells such as macrophages makes them good candidates for useas gene transfer tools. HIV vectors facilitate transduction of quiescenthuman hematopoietic stem cells (HSCs).

Human hematopoietic stem cells (HSC) are an attractive target for genetherapy of inherited hematopoietic disorders as well as other acquireddisorders because these cells have the ability to regenerate the entirehematopoietic system. For example hematopoietic stem cells canregenerate monocytic cells which are known to be involved in humanimmunodeficiency virus-1 (HIV-1) pathogenesis.

Despite more than 15 years of research in the field of gene therapyusing hematopoietic stem cells, the major hurdle remains the inabilityto efficiently and stably insert genes into these cells. Retroviralvectors based upon the Moloney murine leukemia virus (MLV) have beenused most extensively, but yield relatively low gene transfer intopluripotent human HSC and gene expression which is often unsatisfactory.

Recently, attempts of genetic modification of hematopoietic stem cellswith genes that inhibit replication of HIV-1 are aimed, for thedevelopment of monocytes resistant to HIV-1 infection (Kohn et al.1999).

Theoretically, insertion of a gene capable of conferring resistance toHIV-1 into hematopoietic stem cells would result in that gene beingpresent in the descendant mature monocytes and other HIV-1 susceptiblecells Thus, the use of a promoter which is active in monocyte cells suchas the promoter of IL-18BP or a fragment thereof, for HIV-1 gene therapyis advantageous.

Gene therapy of most blood genetic disorders (e.g. SCID chronicgranulomatous disease, thalassemia etc.) requires ex vivo gene transferinto transplantable, self renewing HSCs and regulation of transgeneexpression in one or more cell lineages. Correction of disordersaffecting a specific progeny of HSCs (e.g. hemoglobinopathies orthalassemias, HIV-Infection) requires restricting expression oftherapeutic gene in cell lineage specific fashion (Iotti et al. 2002).In these cases, transcriptional targeting of the transferred gene ismandatory. Gene expression in different cell types is dependent on therelative strength of the promoter used. However, most preclinicalstudies carried out so far have relied on the use of viral, constitutivepromoters to drive transgene expression. For example in HIV-1 vectoruses an internal CMV promoter and the murine CMV promoter the murineretroviral vector LTR. Appropriate transgene regulation in the frameworkof a retroviral vector is a difficult task, due to transcriptionalinterference between the viral long terminal repeat (LTR) and internalenhancer-promoters and genetical instability of complex regulatorysequences. In the present invention the promoter of IL-18BP which isknown to drive transcription in mononuclear cells is used to drivetransgene expression in HSCs, the precursor of such mononuclear cells.

Genetic modification of hematopoietic stem cells with “anti HIV genes”could lead to development of lymphocytes and monocytes resistant to HIVinfection after transplantation. HSC of HIV-1 infected patients can berecovered, CD34+ cells isolated, transduced in vitro with a retroviralvector carring an HIV-1 inhibitory protein under the control of theIL-18BP promoter (instead of the retroviral promoter) and reinfusingthese cells into these patients (Kohn et al. 1999).

The most commonly used source of HSC is peripheral blood hematopoieticstem cells (PBSC), which have largely replaced bone marrow in thesetting of autologous transplantation (Gale et al. 1992 and Kessinger etal. 1991). PBSC are mobilized from the bone marrow into the peripheralcirculation by administration of factors such as G-CSF or GM-CSF for 3-5days and can then be collected by leukapheresis. Several studies haveshown that engraftment occurs faster when transplanting peripheral bloodstem cells instead of bone marrow (Henon et al 1992 and Chao et al.1993). The clonogenic progenitor cells contained in G-CSF-mobilized PBSCare quite susceptible to retroviral-mediated gene transfer, whereas thetransduction rate of long-term reconstituting stem cells in PBSC is nobetter than bone marrow (Breni et al. 1992, Cassel et al. 1993, Dunbaret al. 1995). It has been shown that HIV-1 infected subjects can havesuccessful mobilization and collection of G-CSF-mobilized PBSC withoutany increase in endogenous HIV-1 levels, at least during early stages ofdisease (Junker et al. 1997 and Slobod et al. 1996).

Another source of hematopoietic stem cells is umbilical cord blood (UCB)which has been shown to be susceptible to retroviral transduction,potentially even more so than bone marrow cells (Moritz et al. 1993 andHao et al. 1995). Use of UCB cells HSC could be particularly beneficialfor HIV-1 infected neonates. Since transmission is mostly perinatal, theumbilical cord blood should contain normal numbers and function ofhematopoietic stem cells, which may be diminished in the bone marrow ofHIV-1 infected children and adults (Kearns et al. 1997).

A large number of synthetic genes have been developed which can suppressHIV-1 replication (“anti-HIV-1 genes”), including: antisense, ribozymes,dominant-negative mutants (e.g. RevM10), RNA decoys, intracellularantibodies to prevent expression of viral proteins or cellularco-receptors, etc. (Veres et al. 1996, Zhou et al. 1994, Couture et al1996, Malim et al. 1989, bahner et al 1993 and Sullenger et al. 1990,Lee et al. 1994, Marasco et al 1997 and Chen et al. 1997). In manycases, these anti-HIV-1 genes have been shown in model systems to beable to significantly suppress the replication of HIV-1 and in somecases even limit virus entry into cells (36, 39-44). If essentially 100%of a patient's HSC and the resultant monocytic cells could be madeincapable of supporting HIV-1 replication, it is likely that decreasedviral burdens would result. Theoretically, active inhibition of HIV-1replication in 99.9% of the susceptible cells would be required toproduce a 3-log reduction in virus load, an effect often produced byhighly-effective anti-retroviral therapy. However, with the limitedcapabilities to effectively transduce high percentages of humanhematopoietic stem cells, it is not currently possible to protect themajority of susceptible cells. An alternative mechanism for efficacy isbased on the possibility that cells engineered to be incapable ofsupporting active HIV-1 replication may be protected from viral-inducedcytopathicity and thus have a selective survival advantage compared tonon-protected cells. In that case, a modest number of protected cellsmay comprise an increased percentage of all monocytes, leading to somepreservation of immune function.

The present invention also relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a virus comprisinga sequence of the present invention corresponding to the IL-18BPpromoter region or promoter operably linked to a gene of interestencoding for a suitable drug. These compositions may be used preferablyfor targeting a drug to tissues in which the levels of IFNγ areelevated.

Having now described the invention, it will be more readily understoodby reference to the following examples that are provided by way ofillustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1

Basal Expression of IL-18BP in Monocytes.

IL-18BP MrRNA is detected in leukocytes, colon, small intestine,prostate and particularly in spleen cells (Novick et al. 1999). Spleencells consist of macrophages, lymphocytes, and plasma cells withadditional cells derived from the circulation.

In order to determine expression of IL-18BP protein in cells, a specificELISA test was used (Example 12). Human peripheral blood mononuclearcells (PBMC) were found to constitutively produce IL18BP (0.7-1.5ng/ml). U-937 cells, a cell line derived from malignant cells obtainedfrom the pleural effusion of a patient with histiocytic lymphoma, didnot express any IL-18BP. U-937 cells can be induced to terminalmonocytic differentiation by treatment with phorbol esters. A basalIL-18BP expression of 0.07±0.01 ng/ml was detected only afterdifferentiation of the cells into macrophage-like cells by stimulationwith TPA (10 ng/ml). These results show that IL-18BP is constitutivelyproduced in monocytes and macrophages.

Example 2

Induction of IL-18BP Expression in Various Different Cells.

It has been previously reported that IFNγ induced IL-18BP mRNA andprotein in various cell lines such as a keratinocyte cell line, a coloncarcinoma cell line and in primary renal mesangial cells (Muhl et al.2000). The induction of IL-18BP in various human cell lines and inperipheral blood mononuclear cells (PBMC) by IFNγ and other cytokineswas studied. IFNγ induced IL-18BP expression (see Example 11 formonitoring mRNA and Example 12 for ELISA) in a dose-dependent manner,exhibiting an EC₅₀ at 50 U/ml in WISH and HepG2 cells (FIG. 2 A and B).IL-18BP apparently accumulated in the culture supernatants of WISHcells, as its concentration was higher at 48 h compared with 24 h (FIG.2 A).

Human peripheral blood mononuclear cells (PBMC) constitutively producedIL-18BP (0.7-1.5 ng/ml), and treatment with IFNγ (100 U/ml) increasedthe level of IL-18BP by 1.7±0.1 and 2.1±0.3 fold at 24 and 48 h,respectively (p<0.05, n=4). No effect on IL-18BP production was seenupon pre-treatment of the PBMC with TPA.

IL-18BP induction by IFNγ was tested in the U937 cell line. IFNγ did notinduce IL-18BP in undifferentiated U937 cells, however, followingdifferentiation with phorbol ester (TPA, 10 ng/ml) into macrophage-likecells, a basal level of IL-18BP (0.07±0.01 ng/ml) was obtained, andincreased by 4.6±0.05 fold upon induction with IFNγ(100 U/ml, 24 h),further increasing at 96 h (not shown).

The effect of other cytokines, such as IFNα2, IFNβ, IL-1, IL-6, IL-12,IL-18 and TNFα, on IL-18BP expression was tested in HepG2 cells (FIG.2B). The results obtained following incubation of the cells with thedifferent cytokines in the presence or the absence of IFNγ (FIG. 1 PNASweb) show that IFNα2, IFNβ, IL-1, IL-6, IL-12, IL-18 and TNFα did notinduce IL-18BP alone. However, in HepG2 cells IL-6 and TNFα actedsynergistically with IFNγ, providing a statistically significantincrease of IL-18BP.

These results indicate that IL-18BP can be induced by IFNγ, in monocytesand in many different cells. Induction of IL-18BP by IFNγ is furtherenhanced by the addition of IL-6 and TNFα.

Example 3

The IL-18BP Gene is Transcriptionally Regulated by IFNγ, Requiring DeNovo Protein Synthesis.

In order to check whether the induction of IL-18BP mRNA by IFNγ is atthe transcriptional level, the effect of interferon on HepG2 and Wishcells was measured in the presence of a translation inhibitor,Actinomycin D (FIG. 3A). Increase in IL-18BP mRNA levels were detectableby semi-quantitative RT-PCR after 3 h of treatment with IFNγ in HepG2cells and only after 5 h in Hakat and WISH cells. Pre-treatment of HepG2and WISH cells with Actinomycin D prior to IFNγ stimulation abolishedthe expression of IL-18BP mRNA at various time points, indicating thatIFNγ stimulates de-novo mRNA synthesis.

Accumulation of IL-18BP was apparent 24 h and later following IFNγtreatment, supporting a dependence of IL-18BP expression on precedinginduction of proteins, e.g. transcription factors, by IFNγ. Therefore inorder to confirm such hypothesis, a protein inhibitor, cycloheximide,was further employed to test whether induction of IL-18BP mRNA by IFNγrequires de-novo synthesis of proteins. The results summarized in FIG.3B show that pre-treatment of the cells with cycloheximide abolished theinduction of IL-18BP mRNA. This result indicates that de novo proteinsynthesis is essential for IL-18BP gene activation by IFNγ.

Example 4

Defining the Transcription Start Site of IL-18BPa and its PromoterRegion, in Order to Map the IL-18BP Promoter.

In order to study the IL-18BP promoter region, it is required first tospecifically locate the transcription start site.

The transcription start site of human IL-18BPa mRNA was determined by 5′RACE (RACE Example 14). Only one PCR product, corresponding to IL-18BPa,the most abundant splice variant, was obtained by 5′ RACE. DNA sequenceanalysis of this product revealed the transcription start site and anadditional exon of 50 bp following the transcription start site at the5′-end of human IL-18BPa mRNA, corresponding to positions 785-835 of thegenomic IL-18BP DNA (can be found in the Entrez pubmed nucleotidedatabase, accession No. AF110798). Accordingly, a new exon-intron mapwas generated by comparing the genomic DNA with the mRNA to which thenew 5′ exon was added. (See FIG. 4).

Having the transcription start site of IL-18BPa (base 1), the humangenomic DNA upstream of base 1 (chromosome 11q clone:RP11-757C15,Accession No AP000719.4 nucleotides upstream of base 152,178)corresponding to the IL-18BP promoter region could be further analysed.Comparison of this DNA to the expressed sequence tag (EST) database atNCBI by the BLAST program revealed an upstream gene at the +strand,coding for a Zinc-finger protein (Accession No. AK001961). The depositedmRNA sequence of this Zn finger protein was further elongated by the“Instant RACE” program (www.LabOnWeb.com), which scanned an extensivecollection of human ESTs. The program placed the 3′ end mRNA of the Znfinger protein at nucleotide 150,517 of the genomic clone RP11-757C15,thereby limiting the potential upstream regulatory sequence of theIL-18BPa to 1661 bases upstream of base 1.

Example 5

Exploring the Minimal Promoter, Upstream of the IL-18BP Gene, Capablefor Promoting Constitutive Expression of a Heterologous Gene.

In order to find the minimal DNA fragment, upstream of the IL-18BP gene,capable of directing expression of an hexogenous gene such as theluciferase reporter gene, a vector containing up to 1601 bpcorresponding to the DNA sequence upstream of base 1 and including 50 bpdownstream of the transcription strat site (SEQ ID NO: 5) and vectorshaving truncated forms of this DNA (FIG. 5A) fused to the luciferasegene were generated (Example 15). Luciferase activity in human HepG2cells (a human hepatocellular carcinoma line) transfected with a vector(pGL3(1601)) comprising the 1601 bp upstream DNA was 10.3±0.9 foldhigher than that obtained with the empty pGL3 vector. Such activity wasnot observed when the same DNA was inserted in the opposite orientation(pGL3(-1601). This result demonstrated that the 1601 bp DNA upstream ofbase 1 has basal promoter activity. Sequence examination of this 1601 bpDNA fragment revealed that it does not include a TATA box element, buthad several GC-rich domains near the transcription start site at bases-3 to -9, -39 to 48 and -122 to -132. Analysis of the 1601 bp DNAsequence by the program TFSEARCH identified a gamma-activated sequence(GAS) at bases -24 to -32 (FIG. 1). Further analysis revealed an IRF1,2response element (IRF-E) spanning bases -57 to -69 and two C/EBPβresponse elements (C/EBP-E) at bases -309 to -322 and -621 to -634.

A series of luciferase reporter vectors with progressive truncations atthe 5′ end of the 1601 bp fragment were tested. The results summarizedin FIG. 5A show that all constructs, including pGL3 (122), containingonly the IRF and GAS elements, were at least as effective as pGL3(1601)in supporting basal promoter activity.

These results revealed that the 122 bp fragment (SEQ ID NO:3) comprisingthe IRF and GAS elements are sufficient for promoting basal activity ofan heterologous gene (FIG. 5A).

Example 6

Exploring the Minimal Promoter, Upstream of the IL-18BP Gene, Capablefor Promoting Inducible Expression of a Heterologous Gene.

In order to identify the minimal DNA fragment, upstream region of theIL-18BP promoter, capable of promoting IFNγ induced luciferaceexpression, the truncated DNA vectors from the preceding example weretested in transfected HepG2 cells in the presence of IFNγ (FIG. 5B fortransfections see example 16).

The results summarized in FIG. 5B show that after 24 h IFNγ increasedthe luciferase activity by 33 fold over the basal expression level inthe vector including only IRF-E and GAS elements (pGL3(122) vector).This result demonstrates that the IRF-E-GAS pair alone can mediateheterologous gene induction by IFNγ. Inclusion of C/EBP-E1 and 2elements (pGL3(656)) significantly increased the induction of luciferaseactivity by IFN-γ to 88 fold over the basal activity, demonstrating theimportance of these elements in inductivity by IFNγ. In contrast,inclusion of an additional upstream DNA to such insert (pGL3(1106))abolished the induction of luciferase activity above its basal level.This result suggested that a silencer element resides within bases -656to -1106 (Upstream of the second C/EBP-E1 element). It was demonstratedthat three AP1 response elements are present within the silencer regionand that c-Jun binds to, and is involved in silencing of the IL-18BPgene through all of such three AP-1 response elements.

Further extension of the promoter by 88 bases upstream the silencer(pGL3(1272)) restored the response to IFNγ, suggesting that an enhancerelement resides in bases -1106 to -1272, and its activation by IFNγsuppresses the effect of the neighboring silencer. Further extension ofthe sequence did not affect basal or IFNγ-induced activity, suggestingthat all upstream regulatory sequences were located within bases -1 to-1272 (SEQ ID NO:1).

From all of the constructs tested the inductivity of the pGL3(656) isthe highest, indicating that this DNA fragment contains the optimalinducible promoter of IL-18BP.

The results show that the minimal inducible promoter is located 122 bpupstream of the transcription start site (SEQ ID NO:3) containing theIRF-E and GAS elements, wherein the maximal and optimal induciblepromoter is located 656 bp upstream of the transcription start site (SEQID NO: 2) containing in addition to the IRF-E and GAS elements, twoC/EBPβ elements.

Example 7

Involvement of IRF-1 in IL-18BP Expression In-Vivo.

To explore the involvement of IFR-1, the binding site of which was foundto be in the promoter of IL-18BP, in IL-18BP expression the expressionof IL-18BP in IRF-1 defficient mice was studied.

The levels of IL-18BP were measured in IRF-1-deficient mice (Jacksonlaboratories, Bar Harbor Me.) before and after administration of murineIFNγ and compared to those in control C57B 1/6 mice (FIG. 6). Basalserum IL-18BP in control C57B1/6 mice was 9.1±1.9 ng/ml and wassignificantly increased by IFNγ to 22.4±2.2 ng/ml. In contrast, serumIL-18BP in IRF-1-deficient mice was below the limit of detection andincreased to only 0.7±1.15 ng/ml with IFNγ. This result confirmed theimportance of IRF-1 as a mediator of basal as well as IFNγ-inducedexpression of IL 18BP.

Example 8

Detection of the Transcription Factors Binding to the IL-18BP PromoterUnder Inductive Conditions.

Electrophoretic mobility shift assays (EMSA Example 18) were employed toidentify protein-DNA interactions among the various response elementswithin the IL-18BP promoter. Labelled ds DNA probes corresponding tobases -33 to -75 (containing the IRF-E) and -8 to -55 (containing theGAS) were allowed to bind with nuclear extracts of control andIFNγ-treated cells. A complex of the IRF-E-containing probe and nuclearprotein(s) was apparent following incubation of cells for 1 h with IFNγand maximal response was seen at 3 h (FIG. 7 A, lanes 1-5). As expected,addition of antibodies to IRF-E caused a “super-shift”, whereas controlanti-signal transducer and activator of transcription 1 (STAT1)antibodies had no effect (data not shown). In contrast with IRF-E, theGAS-containing probe was constitutively associated with a protein (FIG.7 A, lane 6) and this complex was enhanced upon induction of cells withIFNγ for 3 to 6 h (lanes 7, 8). GAS is expected to bind the IFNγ-inducedSTAT1 dimer. Nevertheless, the complex was not affected by antibodies toSTAT1 (lane 10), suggesting that the IFNγ-induced STAT1 dimer was notassociated with this GAS. The same negative result was obtained withnuclear extracts of cells treated with IFNγ for only 15 or 30 min (datanot shown). Surprisingly, this complex was abolished by antibodies toC/EBPβ (lane 9) and was super shifted with antibodies to IRF-1 (lane10). Hence the GAS-containing DNA probe appears to bind C/EBPβ despitelack of a consensus C/EBPβ E.

The results obtained with EMSA indicate that upon induction with IFNγ,IRF-1 binds to the IRF-E element in the IL-18BP promoter. In addition, acomplex comprising IRF-1 and C/EBPβ is formed and binds to the GASelement.

Example 9

Exploring the Role of the IRF-1-C/EBPβ Complex in IL-18BP Induction.

To further study the role of IRF-1 and C/EBPβ in IL-18BP gene induction,IL 18BPa mRNA was measured by semi-quantitative PT-PCR followingoverexpression of IRF-1 and C/EBPβ by employing transfection ofexpression vectors (Example 14, FIG. 7 B). Over-expression of eithertranscription factor or a combination of both factors in HepG2 cells didnot induce IL-18BP mRNA. This result suggested that additionalIFNγ-induced factors are required for activation of the IL-18BP gene.Transfection of the cells with either one of the expression vectorsfollowed by their induction with IFNγ actually reduced IL-18BP mRNAcompared with IFNγ alone. In contrast, co-expression of the twotranscription factors increased the induction of IL-18BP mRNA by IFNγ.This result suggested that IRF-1 and C/EBPβ must be present at a certainratio, possibly forming a complex within the transcription initiationcomplex. To further study the possible interaction between IRF-1 andC/EBPβ a titration of luciferase activity by co-transfecting cells withpGL3 (1272), a fixed amount of an IRF-1 expression vector and varyingamounts of C/EBPβ expression vector was performed. Similarly, luciferaseactivity when the C/EBPβ vector was kept constant and with varyingamounts of the IRF-1 vector was measured. In both cases a bell-shapeddose-response curve was obtained, suggesting that optimal IL-18BPinduction requires a fixed molar ratio between these two transcriptionfactors (FIG. 7 C).

Immunoprecipitation studies were carried out in order to confirm thephysical association between IRF-1 and C/EBPβ (Example 19, FIG. 7 D).Immunoprecipitation (ip) followed by immunoblotting (ib) of nuclear andcytoplasmic proteins (Example 15) from IFNγ-treated cells withantibodies to C/EBPβ revealed that C/EBPβ is constitutively expressed inHepG2 cells and translocates to the nucleus in response to IFNγ (upperpanel). In contrast to C/EBPβ which is not induced by IFNγ, ip and ib ofcell extracts with antibodies to IRF-1 revealed that IFNγ induces theexpression of IRF-1. But similar to C/EBPβ, upon IFNγ induction, IRF-1is translocated to the nucleus (middle panel). Ip with antibodies toC/EBPβ, followed by ib with antibodies to IRF-1 revealed the presence ofa stable IRF-1-C/EBPβ complex in the nuclear fraction (lower panel).These results confirm the formation of the IRF-1-C/EBPβ complex uponIFNγ induction and is the first demonstration of the existence of such acomplex between these two transcription factors. Thus upon IFNγinduction, the proximal GAS-containing sequence and its adjacent IRF-Ebind the complex of C/EBPβ and IRF-1.

Example 10

Exploring the Role of the C/EBP-Es in the IL-18BP Promoter Activity.

The two C/EBPβ sites at positions -309 to -322 and -621 to -634 do nothave an adjacent IRF-E. Indeed, EMSA (Example 18) of a probecorresponding to the C/EBPβ sites at positions -309 to -322 revealed aretarded band (filled arrowhead) that was super shifted with antibodiesto C/EBPβ (open arrowhead) but not with antibodies to IRF-1 (FIG. 8 A).Hence, it was concluded that this site binds C/EBPβ and not its complexwith IRF-1. Furthermore, this band was generated with a nuclear extractof un-induced HepG2 cells that constitutively express C/EBPβ but lackIRP-1. In fact, IFNγ did not increase the expression of C/EBPβ in thesecells (FIG. 8 D) and consequently it did not increase the intensity ofthe retarded band (FIG. 8 A). Similar results were obtained with themore distal C/EBPβ site (data not shown).

The results show that the C/EBP transcription factor unlike the IRF-1,is constitutively expressed and not induced by IFNγ and that in additionof binding to IRF-1 and to GAS, it binds to both of the C/EBP elementspresent in the IL-18BP promoter.

Example 11

Studying the Role of the Enhancer in the Expression of IL-18BP.

The regulatory role of the distal enhancer was studied by EMSA (Example18) with a 192 bp DNA probe, corresponding to nucleotides -1081 to-1272. Nuclear extract of control HepG2 cells formed a complex with thisprobe (FIG. 8 B, filled arrowhead). Upon treatment of the cells withIFNγ, the complex was more intense and somewhat more retarded. Asuper-shift of this complex with antibodies directed against IRP-1,C/EBPβ and cFos was then performed. Of these, only anti IRF-1 elicited asuper-shift (FIG. 8 B, empty arrowhead). An unlabelled dsDNAcorresponding to nucleotides -1083 to -1174 did not compete with theradiolabeled probe, indicating that the nuclear proteins were bound toresidues -1175 to -1272. Since IRF-E was identified only in the proximalregion, this result suggests that the distal enhancer was probablyassociated with the proximal IRF-E.

These results indicate that the distal enhancer interacts with the basalpromoter through IRF-1.

Example 12

ELISA for IL-18BP.

Human IL-18BP was measured by a double antibody ELISA as described(Novick et al 2001). Mouse IL-18BP was measured by a double antibodyELISA using rabbit antigen affinity-purified polyclonal antibody tomurine IL-18BP and biotinylated antibody that were obtained fromCytolab, Israel.

Example 13

RNA Isolation and Reverse Transcription (RT)-PCR.

Following treatment in serum-free medium, HepG2, and WISH cells (10⁶)were harvested at the indicated times and total RNA was extracted usingTRI reagent. cDNA was prepared using random hexamers and SuperscriptII(Invitrogen™, Leek, The Netherlands) according to the manufacturer'sinstructions. PCR was performed with the following primers: humanIL-18BP, 5′ CACGTCGTCACTCTCCTGG and 5′ CGACGTGACGCTGGACAAC; human IRF-15′ GACCCTGGCTAGAGATGCAG and 5′ GAGCTGCTGAGTCCATCAG; human βActin 5′GTGGGGCGCCCCAGGCACCA and 5′ CTCCTTAATGTCACGCACGATTTC. Amplificationswere done by initial denaturation (92° C., 2 min), 28 cycles ofdenaturation (92° C., 45 sec.), annealing (62° C., 1 min) and extension(72° C., 1.5 min), and final extension (72° C., 10 min). The resultingPCR products were resolved by agarose (1%) gel electrophoresis.

Example 14

Rapid Amplification of 5′ cDNA Ends (5′ RACE).

5′ RACE was performed with a 5′ RACE System (GIBCO BRL) according to themanufacturer's instructions. Briefly, total RNA from IFNγ-treated WISHcells was reverse-transcribed (Example 13) with a primer complementaryto nucleotides 89-70 of IL-18BPa mRNA (GenBank Accession No. AF110799)followed by tailing of the newly synthesized ends with an anchor DNA.PCR was then performed with a forward primer complementary to the anchorDNA and a nested reverse primer complementary to nucleotides 31-11 ofIL-18BPa mRNA. The PCR products were then subcloned and subjected to DNAsequence analysis.

Example 15

Plasmids and Cloning.

The entire putative IL-18BPa promoter region of 1601 bp was obtained byPCR of genomic DNA using a sense primer (S4753.pgl) containing a Kpn Isite (5′ CTATATGGTACCCACCCTTCCTTTTACTTTTTCC) and reverse primer (R1exA)containing Nhe I site (5′ TATCGCTAGCCAGTCACACAGGGAGGCAGT). The PCRproduct was cloned into pGEM-T Easy vector (Promega, Madison, Wis.) andverified by DNA sequence analysis. A Kpn I-Nhe I fragment was isolatedfrom the pGEM-T Easy clone and ligated into pGL3-Basic vector (Promega)using Rapid DNA Ligation Kit (Roche) to give pGL3(1601). A series of5′-truncated reporters (pGL3(1454), pGL3(1274), pGL3(1106), pGL3(656),pGL3(280) and pGL3(122) was similarly prepared with the same reverseprimer and with the following sense primers, respectively:

S334.pgl 5′: CTATATGGTACCCATGAACTAGACACCTAGAG; S415.pgl 5′:CTATATGGTACCCTACAAGAAGTTTGAGATCA; S501.pgl 5′:CTATATGGTACCCAGCCGTTGCACCCTCCCAATCAC; 1exD pgl5′ CTATATGGTACCGTCTTGGAGCTCCTAGAGG; S504.pgl5′ CTATATGGTACCCACCAAAGTCCTGACACTTG and S139.pgl5′ TTGGTACCCACTGAACTTTGGCTAAAGC..All PCR products were also cloned into pGL3-Basic vector in the oppositeorientation to serve as controls.

Example 16

Transient Transfection Assays.

HepG2 or WISH cells in 6 well plates (0.5×10⁶/well) were transfectedusing FuGENE 6 and the indicated luciferase reporter vector (0.5μg/well) and pSV40 βGAL (0.2 μg/well, Promega) according tomanufacturer's instructions. In some cases co-transfection was done withthe following expression vectors: poDNA3-IRF-1 (0.07-1.5 μg/well, kindlyprovided by Dr. B. Levy, Technion, Israel); pcDNA3-C/EBPβ (0.5-2.5μg/-well, kindly provided by Dr. D. Zipori, Weizmann Institute ofScience). After 16 h cells were treated with either IFNγ (100 U/ml),IL-6 (150 U/ml), TNFα (10 ng/ml) or their indicated combinations inserum-free medium for 24 h. Cells were then collected, lysed andLuciferase activity was measured. All results were normalized againstβ-galactosidase activity,

Example 17

Preparation of Nuclear and Cytoplasmic Extracts.

Cells were washed 3×with ice-cold phosphate buffered saline (PBS) andimmediately frozen in liquid nitrogen. Cell pellets were re-suspended infour packed cell volume of cytoplasmic buffer (10 mM Hepes, pH 7.9, 10mM NaCl, 0.1 mM EDTA, 5% (by vol.) glycerol, 1.5 mM MgCl₂, 1 mMdithiothreitol (DTT), 0.5 mM PMSF, 50 mM NaF, 0.1 mM Na₃VO₄, 2 mM EGTA,10 mM EDTA, 10 mM Na₂MoO₄, 2 μg/ml each of leupeptin, pepstatin andaprotinin). The lysate was centrifuged (3000×g, 10 min.) and thesupernatant containing the cytoplasmic proteins was collected. Thepellet was re-suspended in 2.5 packed cell volumes of nuclear buffer(identical to cytoplasmic buffer except that NaCl was increased to 0.42M). Nuclear debris was removed by centrifugation (15,000×g, 20 min. 4°C.), aliquots of the supernatant were frozen in liquid nitrogen andstored at −80° C. Protein concentration was determined by a BCA Proteinassay reagent kit (Pierce, Rockford USA) using bovine serum albumin as astandard.

Example 18

Electrophoretic Mobility Shift Assays.

Ds oligonucleotides corresponding to selected response elements (10pmol) were labeled with [³² P]3 ATP by polynucleotide kinase (NewEngland Biolabs). Nuclear extracts (5 μg protein) were pre-incubated (15min., 0° C.) together with poly(dI-dC) (Amersham Pharmaciabiotechnology) in 20 μl-EMSA Buffer (20 mM Hepes pH 7.5; 5 mM MgCl₂, 2mM EDTA, 5 mM DTT and 5% (by vol.) glycerol). A labeled probe (3×10⁴cpm) was then added and incubation continued for an additional 30 min.at room temperature. For super-shift assays samples were incubated withthe indicated antibodies (4 μg, 1 h at 0° C.) prior to addition of theprobe. A 200 fold excess of wild type and mutated competitors were addedtogether with the relevant probe. Reaction mixtures were thenelectrophoresed in 5% non-denaturing polyacrylamide gels. Gels werevacuum dried and autoradiographed overnight at −80° C.

Example 19

Immunoprecipitation (ip) and Immunoblot (ib) Analysis.

Nuclear or cytoplasmic protein extracts (80 μg) were incubated with 6 μgof the indicated polyclonal antibodies overnight at 4° C. andimmunoprecipitated with Protein G Sepharose beads (Pharmacia) for 1 h atroom temperature. The beads were then boiled in SDS-PAGE sample buffercontaining 10% DTT and the supernatant resolved by SDS-PAGE (10%acrylamide) under reducing conditions. The gel was then blotted onto anitrocellulose membrane and proteins detected with the indicatedantibodies. Immune complexes were identified by Super Signal™ (Pierce)detection kit.

Example 20

Preparation of CHO r-hsLDLR Using the IL-18BP Promoter.

Stable recombinant CHO cells expressing human soluble LDLR are generatedby co-transfection of CHO-DUKX cells lacking the dihydrofolate reductase(DHFR) gene (Urlaub, G. et al., 1980) with two expression vectors: onecontaining the N-terminal ligand-binding domain of the LDLR, beginningat amino acid residue Asp (+4) up to Glu 291 (+291), and pDHFR,containing the murine gene for DHFR, DHFR controlled by the early SV40promoter and sLDLR gene by the IL-18BP promoter (SEQ ID NO:2) andtranscription termination elements of the SV40 early region.Transfection is performed by cationic liposomes using LipofectAmine(Gibco BRL), according to the protocol described by the manufacturer.Seventy-two hours after transfection cells are transferred to aselective medium lacking deoxy and ribonucleosides and supplemented with10% dialysed FCS. Cells expressing DHFR activity are able to formcolonies, which are isolated by lifting the cells with trypsin-soakedpaper discs The cells are grown and screened for r-hsLDLR activity. Thetransfected cells are then subjected to gene amplification by MIX,followed by subcloning and selection of the stable producer clones.

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1. An isolated DNA sequence comprising a functional human IL-18BPpromoter sequence comprising the nucleotide sequence of SEQ ID NO: 1 andfurther comprising SEQ ID NO: 5 operably linked to the 3′ end of SEQ IDNO:1.
 2. The isolated DNA sequence according to claim 1, operably linkedto an intron.
 3. The isolated DNA sequence according to claim 2, whereinthe intron consists of the first intron of IL-18BP.
 4. The isolated DNAsequence according to claim 1, further comprising a gene operativelylinked to the isolated DNA sequence.
 5. The isolated DNA sequenceaccording to claim 4, wherein the gene encodes IL-18BP.
 6. The isolatedDNA sequence according to claim 4, wherein the gene encodes aheterologous protein.
 7. The isolated DNA sequence according to claim 6,wherein the heterologous protein is a luciferase protein.
 8. Theisolated DNA sequence according to claim 6, wherein the heterologousgene encodes a protein selected from an interferon-beta, a TNF, anerythropoietin, a tissue plasminogen activator, a granulocyte colonystimulating factor, a manganese-superoxide 41 dismutase, animmunoglobulin, an immunoglobulin fragment, a growth hormone, an FSH, anhCG, an IL-18, an hsLDLR and a TNF receptor binding protein.
 9. A vectorcomprising the DNA sequence according to claim
 1. 10. An isolated hostcell comprising the vector according to claim
 9. 11. The isolated hostcell according to claim 10, which is a mammalian cell.
 12. The isolatedhost cell according to claim 11, selected from the group consisting ofCHO, WISH, HepG2, Cos, CV- 1.HeLA, and Hakat U937 cells.
 13. Arecombinant virus vector which comprises a portion of the virus genomicnucleic acid, a DNA fragment comprising a gene of interest and a DNAfragment comprising the DNA sequence according to claim 1, operablylinked to the gene of interest.
 14. The recombinant virus vectoraccording to claim 13, wherein the gene of interest encodes a proteinselected from an interferon-beta, a TNF, an erythropoietin, a tissueplasminogen activator, a granulocyte colony stimulating factor, amanganese-superoxide dismutase, an immunoglobulin, an immunoglobulinfragment, a growth hormone, an FSH, an hCG, an IL-18, an hsLDLR and aTNF receptor binding protein.
 15. The recombinant virus vector accordingto claim 13, wherein the virus is an adeno-associated virus.
 16. Apharmaceutical composition comprising the isolated DNA sequence ofclaim
 1. 17. An isolated DNA sequence comprising a functional humanIL-18BP promoter comprising the nucleotide sequence of SEQ ID NO: 1 andfurther comprising SEQ ID NO: 5 operably linked to the 3′ end of SEQ IDNO:1 and wherein the promoter is mutated at one or more AP1 sitespresent in the silencer element present in SEQ ID NO:
 1. 18. Theisolated DNA sequence according to claim 17, further comprising anintron.
 19. The isolated DNA sequence according to claim 18, wherein theintron consists of the first intron of IL-18BP.
 20. The isolated DNAsequence according to claim 17, further comprising a gene operativelylinked to the isolated DNA sequence.
 21. The isolated DNA sequenceaccording to claim 20, wherein the gene encodes IL-18BP.
 22. Theisolated DNA sequence according to claim 20, wherein the gene encodes aheterologous protein.
 23. The isolated DNA sequence according to claim22, wherein the heterologous protein is a luciferase protein.
 24. Theisolated DNA sequence according to claim 22, wherein the heterologousgene encodes a protein selected from an interferon-beta, a TNF, anerythropoietin, a tissue plasminogen activator, a granulocyte colonystimulating factor, a manganese-superoxide 41 dismutase, animmunoglobulin, or a fragment thereof, a growth hormone, an FSH, an hCG,an IL-18, an hsLDLR and a TNF receptor binding proteins.
 25. A vectorcomprising the DNA sequence according to claim
 17. 26. An isolated hostcell comprising the vector according to claim
 25. 27. The isolated hostcell according to claim 26, which is a mammalian cell.
 28. The isolatedhost cell according to claim 27, selected from the group consisting ofCHO, WISH, HepG2, Cos, CV- 1.HeLA, and Hakat U937 cells.
 29. Arecombinant virus vector which comprises a portion of the virus genomicnucleic acid, a DNA fragment comprising a gene of interest and a DNAfragment comprising the DNA sequence according to claim 17, operablylinked to the gene of interest.
 30. The recombinant virus vectoraccording to claim 29, wherein the gene of interest encodes a proteinselected from an interferon-beta, a TNF, an erythropoietin, a tissueplasminogen activator, a granulocyte colony stimulating factor, amanganese-superoxide dismutase, an immunoglobulin, an immunoglobulinfragment, a growth hormone, an FSH, an hCG, an IL-18, an hsLDLR and aTNF receptor binding protein.
 31. The recombinant virus vector accordingto claim 29, wherein the virus is an adeno-associated virus.
 32. Apharmaceutical composition comprising the isolated DNA sequence of claim17.